The process by which nascent proteins are folded into the three-dimensional conformation of their native state will be studied from the standpoint of how chaperones mediate this process on the ribosome on which the protein is synthesized. A cell-free system has been derived from E. coli in which the ribosomal synthesis of many proteins in enzymatically active form is dependent on the addition of the bacterial chaperones (DnaJ, DnaK, GrpE, GroEL, GroES). Fluorescence techniques will be used extensively to study how the individual chaperones mediate the reactions by which the nascent protein is folded during its synthesis in this system. Coumarin, the fluorophore of choice for most experiments, will be incorporated with methionine from N(coumarin-SAc) Met-tRNAf at the amino terminus of the nascent protein during initiation of its synthesis. Changes in fluorescence quantum yield, anisotropy, emission spectrum and lifetime that occur as the nascent protein is extended and folded by the chaperone- mediated reactions will be monitored as will the effect of quenching agents and the availability of the coumarin probe to anticoumarin antibodies and their Fab fragments. The results should provide a model if not the mechanism of the folding steps that lead to the native three-dimensional conformation of the protein. Folding of proteins into their biologically active conformation is fundamental for all living organisms. Development of an understanding of the folding process has been characterized as one of the most important unsolved problems of molecular biology. Knowledge of the reaction mechanisms that are involved will contribute to medicine and biotechnology in a myriad of direct and indirect ways. One especially important aspect concerns protein engineering. Without an understanding of the basic processes and reactions that determine how proteins fold it will be difficult if not impossible to develop a comprehensive theory for the rational design and synthesis of active enzymes and other proteins that may not have counterparts in living cells.